Methods and compositions for treating hiv

ABSTRACT

The invention features nucleic acid constructs encoding chimeric immune T-Cell receptors (CIRs) that are useful for treating HIV in patients. In general, the CIRs contain an extracellular domain which targets HIV or HIV infected cells (e.g., the extracellular domain of CD4), a transmembrane domain, and a cytoplasmic domain for mediating T-Cell activation (e.g. CD3 zeta and/or the partical extracellular domain of CD28). The invention also features the use of host cells expressing CIRs in the treatment of HIV.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/324,050, filed Apr. 14, 2010, which is hereby incorporated byreference.

STATEMENT AS TO FEDERALLY FUNDED RESEARCH

This work was supported by grant number NIH R21, 1R21AI076145-01 fromthe United States National Institutes of Health. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

The invention relates to the field of treating HIV with modifiedT-cells.

In 1984, HIV was shown to be the etiologic agent of AIDS. Since thattime, the definition of AIDS has been revised a number of times withregard to what criteria should be included in the diagnosis. However,despite the fluctuation in diagnostic parameters, the simple commondenominator of AIDS is the infection with HIV and subsequent developmentof persistent constitutional symptoms and AIDS-defining diseases such asa secondary infections, neoplasms, and neurologic disease.

HIV is a human retrovirus of the lentivirus group. The four recognizedhuman retroviruses belong to two distinct groups: the human Tlymphotropic (or leukemia) retroviruses, HTLV-1 and HTLV-2, and thehuman immunodeficiency viruses, HIV-1 and HIV-2. The former aretransforming viruses whereas the latter are cytopathic viruses.

HIV-1 has been identified as the most common cause of AIDS throughoutthe world. Sequence identity between HIV-2 and HIV-1 is about 40%, withHIV-2 being more closely related to some members of a group of simianimmunodeficiency viruses (SIV).

The main cause of the immune defect in AIDS has been identified as aquantitative and qualitative deficiency in the subset of thymus-derived(T) lymphocytes, the T4 population. This subset of cells is definedphenotypically by the presence of the CD4 surface molecule, which hasbeen demonstrated to be the cellular receptor for HIV. Although the T4cell is the major cell type infected with HIV, essentially any humancell that expresses the CD4 molecule on its surface is capable ofbinding to and being infected with HIV.

Previous attempts to treat patients with “designer” T-cells expressingchimeric immune receptors (CIRs) proved unsuccessful. There exists aneed in the art for new therapies for HIV. The present inventionaddresses this issue and offers advantages over previous attemptedtherapies.

SUMMARY OF THE INVENTION

In one aspect, the invention features a nucleic acid construct encodinga chimeric protein that includes (i) an extracellular domain of CD4(e.g., amino acids 1-372 of SEQ ID NO:1) or a fragment thereof, (ii) atransmembrane domain, and (iii) a cytoplasmic domain that includes thecytoplasmic domain of the CD3 zeta chain (e.g., a polypeptide having theamino acids 31-142 of SEQ ID NO:3), or a fragment thereof, and thecytoplasmic domain of CD28 (e.g., a polypeptide having amino acids127-234 of SEQ ID NO:2), or a fragment thereof. In one embodiment, thecytoplasmic domain has the amino acid sequence of SEQ ID NO:10.

In another aspect, the invention features a nucleic acid constructencoding a chimeric protein that includes (i) an extracellular domain ofCD4 (e.g., a polypeptide having amino acids 1-372 of SEQ ID NO:1) or afragment thereof, (ii) a transmembrane domain, and (iii) a cytoplasmicdomain of the CD3 zeta chain (e.g., a polypeptide having the amino acids31-142 of SEQ ID NO:3), or a fragment thereof.

In either of the foregoing aspects, the chimeric protein can be capableof forming a homodimer when expressed in a T-cell, e.g., through theformation of a disulfide bond.

Also in either of the foregoing aspects, the transmembrane domain can bethe transmembrane domain of the CD3 zeta chain (e.g, a polypeptidehaving amino acids 7-30 of SEQ ID NO:3) or the transmembrane/partialextracellular domain of CD28.

Also in either of the foregoing aspects, the chimeric protein caninclude a c-myc tag (e.g., at the N-terminus).

In another aspect, the invention features a vector including any of thenucleic acid constructs described above. This vector can also include anucleic acid construct encoding an siRNA (e.g., against CCR5 or againstTat/Rev).

In yet another aspect, the invention features a host cell (e.g., aT-cell derived from an uninfected patient or T-cell derived from apatient infected with HIV) containing any of the above nucleic acidconstructs or vectors. This host cell can also include a nucleic acidconstruct encoding an siRNA (e.g., against CCR5 or against Tat/Rev).

In another aspect, the invention features a method of treating a patientinfected with HIV (e.g., HIV-1 or HIV-2) by administering a compositionincluding any of the foregoing host cells. In this aspect, the host cellcan be isolated from the patient being treated or from another patient.

By “specifically binds” is meant an extracellular domain whichrecognizes and binds an HIV protein, but that does not substantiallyrecognize and bind other molecules in a sample, e.g., a human bloodsample.

By “treating” is meant ameliorating a condition or symptom(s) of thecondition (e.g., the symptoms of HIV infection). To “treat HIV” orrefers to administering a treatment to a subject infected with HIV toimprove the subject's condition. As compared with an equivalentuntreated control, such amelioration or degree of treatment is at least5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, asmeasured by the subject's HIV viral load.

By “vector” is meant a DNA molecule, usually derived from a plasmid orbacteriophage, into which fragments of DNA may be inserted or cloned. Arecombinant vector will contain one or more unique restriction sites,and may be capable of autonomous replication in a defined host orvehicle organism such that the cloned sequence is reproducible. A vectorcontains a promoter operably-linked to a gene or coding region suchthat, upon transfection into a recipient cell, an RNA or an encodedprotein or is expressed.

By “host T-cell” is meant a cell (e.g., a human T-cell isolated from asubject) into which one or more nucleic acid constructs is introduced.

By “chimeric immune T-cell receptor” or “CIR” is meant a fusion proteinwhich, when expressed in a host T cell, contains an extracellular domainthat specifically binds to a target protein and a cytoplasmic domainthat modulates activation of the host T-cell.

By “CD4 extracellular domain” is meant a polypeptide having theN-terminal region of CD4 that is located outside the cell membrane whenexpressed in a T-cell, e.g., a polypeptide having the amino acidsequence of amino acids 1-372 of SEQ ID NO:1. The term “CD4extracellular domain” is also meant to include any polypeptide fragmentthat binds specifically to gp120 and is substantially identical to aminoacids 1-372 of SEQ ID NO:1 over the length of the polypeptide fragment.

Human CD4 Amino Acid sequence SEQ ID NO: 1LOCUS NP_000607 458 aa linear PRI 18-MAR-2010DEFINITION T-cell surface glycoprotein CD4 precursor [Homo sapiens].ACCESSION NP_000607 VERSION NP_000607.1 GI: 10835167DBSOURCE REFSEQ: accession NM_000616.3 1MNRGVPFRHL LLVLQLALLP AATQGKKVVL GKKGDTVELT CTASQKKSIQ FHWKNSNQIK 61ILGNQGSFLT KGPSKLNDRA DSRRSLWDQG NFPLIIKNLK IEDSDTYICE VEDQKEEVQL 121LVFGLTANSD THLLQGQSLT LTLESPPGSS PSVQCRSPRG KNIQGGKTLS VSQLELQDSG 181TWTCTVLQNQ KKVEFKIDIV VLAFQKASSI VYKKEGEQVE FSFPLAFTVE KLTGSGELWW 241QAERASSSKS WITFDLKNKE VSVKRVTQDP KLQMGKKLPL HLTLPQALPQ YAGSGNLTLA 301LEAKTGKLHQ EVNLVVMRAT QLQKNLTCEV WGPTSPKLML SLKLENKEAK VSKREKAVWV 361LNPEAGMWQC LLSDSGQVLL ESNIKVLPTW STPVQPMALI VLGGVAGLLL FIGLGIFFCV 421RCRHRRRQAE RMSQIKRLLS EKKTCQCPHR FQKTCSPI

By “CD28 cytoplasmic domain” is meant a polypeptide having theC-terminal region of CD28 that is located in the cytoplasm whenexpressed in a T-cell, e.g., a polypeptide having the amino acidsequence of amino acids 127-234 of SEQ ID NO:2. The term “CD28cytoplasmic domain” is also meant to include any polypeptide fragmentthat maintains the ability to modulate activation of T-cells (e.g., asdetermined using the method titled “killing of HIV infected cells bymodified T-cells” below) and is substantially identical to amino acids127-234 of SEQ ID NO:2 over the length of the polypeptide fragment.

Human CD28 Amino Acid sequence: SEQ ID NO: 2LOCUS NP_006130 220 aa linear PRI 11-APR-2010DEFINITION T-cell-specific surface glycoprotein CD28 precursor[Homo sapiens]. ACCESSION NP_006130 VERSION NP_006130.1 GI: 5453611DBSOURCE REFSEQ: accession NM_006139.2   1MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD  61SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP  21PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR 181SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS

By “CD3 zeta” is meant a polypeptide having the amino acid sequence ofSEQ ID NO:3. The term “CD3 zeta” is also meant to include anypolypeptide fragment that maintains the ability to modulate activationof T-cells (e.g., as determined using the method titled “killing of HIVinfected cells by modified T-cells” below) and is substantiallyidentical to SEQ ID NO:3 over the length of the protein fragment.

Human CD3 zeta Amino Acid sequence SEQ ID NO: 3LOCUS NP_000725 163 aa linear PRI 11-APR-2010DEFINITION T-cell receptor zeta chain isoform 2 precursor[Homo sapiens]. ACCESSION NP_000725 VERSION NP_000725.1 GI: 4557431DBSOURCE REFSEQ: accession NM_000734.3 1MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD 61APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP RRKNPQEGLY NELQKDKMAE 121AYSEIGMKGE RRRGKGHDGL YQGLSTATKD TYDALHMQAL PPR

By “small interfering RNA” or “siRNA” is meant an isolated RNA molecule,either single-stranded or double stranded that is at least 15nucleotides, preferably, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, or 35 nucleotides in length and even up to 50 or100 nucleotides in length (inclusive of all integers in between).Preferably, the siRNA is capable of mediating RNAi. As used herein thephrase “mediates RNAi” refers to (indicates) the ability to distinguishwhich RNAs are to be degraded by the RNAi machinery or process. siRNAsare processed from long dsRNAs and are usually double-stranded (e.g.,endogenous siRNAs). siRNAs can also include short hairpin RNAs in whichboth strands of an siRNA duplex are included within a single RNAmolecule. These terms include double-stranded RNA, single-stranded RNA,isolated RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution, and/or alterationof one or more nucleotides.

By “substantially identical” is meant a nucleic acid or amino acidsequence that, when optimally aligned, for example using the methodsdescribed below, share at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity with a second nucleic acidor amino acid sequence. “Substantial identity” may be used to refer tovarious types and lengths of sequence, such as full-length sequence,epitopes or immunogenic peptides, functional domains, coding and/orregulatory sequences, exons, introns, promoters, and genomic sequences.Percent identity between two polypeptides or nucleic acid sequences isdetermined in various ways that are within the skill in the art, forinstance, using publicly available computer software such as SmithWaterman Alignment (Smith and Waterman (1981) J Mol Biol 147:195-7);“BestFit” (Smith and Waterman, Advances in Applied Mathematics, (1981)482-489) as incorporated into GeneMatcher Plus™, Schwarz and Dayhof,Atlas of Protein Sequence and Structure, Dayhof, M.O., Ed (1979)353-358; BLAST program (Basic Local Alignment Search Tool; (Altschul etal. (1990) J Mol Biol 215: 403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X,WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. Inaddition, those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.In general, for proteins or nucleic acids, the length of comparison canbe any length, up to and including full length (e.g., 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine.

Other features and advantages of the invention will be apparent from thefollowing Detailed Description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the structure of the indicated chimericimmune T-cell receptors (CIRs).

FIG. 2 is a diagram showing the organization of an exemplary nucleicacid construct encoding a 2^(nd) generation CIR and siRNA construct.

FIG. 3 is a series of graphs showing the cellular surface expression ofthe indicated CIR. PBMCs were transduced with retrovirus and stainedwith anti-CD4-FITC and anti-CD8-APC antibodies after four days andanalyzed by flow cytometry. % CD8+ cells expressing Hege CIR is 52%,1^(st) generation CIR is 44%, and 2^(nd) generation CIR is 39%. Arepresentative experiment of four is shown.

FIG. 4A is a pair of graphs showing survival of the indicated cell typewhen incubated with HIV-infected CEM-SS cells at the indicated ratio.Transduced PBMCs were co-cultured with HIV-infected CEM-SS cells oruninfected CEM-SS cells at an Effector to Target (E:T) ratio of 1:1 or1:10. Aliquots of cells were taken from the cultures at day three andstained with anti-CD4 and anti-CD8 antibodies as described above. Datashown is % modified cells (CD8+CIR+) at each time point. Arepresentative of two experiments is shown.

FIG. 4B is a pair of graphs showing flow cytometry analysis from daythree for Hege CIR cells. Hege CIR T-cells disappear (Hege+HIV, upperright quadrant) when cultured at an E:T ratio of 1:10. % Modifedcells=(Q2/Q2+Q4) is shown in each plot. (Q2 is upper right quadrant andQ4 is lower right quadrant.)

FIG. 5 is a graph showing survival of Hege CIR cells when incubated withHIV-infected CEM-SS cells at the indicated ratio in the presence andabsence of AZT. Transduced PBMCs were co-cultured with HIV-infectedCEM-SS cells or uninfected CEM-SS cells at an Effector to Target (E:T)ratio of 1:1 or 1:10. Aliquots of cells were taken from the cultures atday three and stained with anti-CD4 and anti-CD8 antibodies as describedfor FIG. 3. Data shown is % modified cells (CD8+CIR+ cells) from oneexperiment.

FIG. 6 is a series of graphs showing expression of the indicated markerson the indicated cells when exposed to HIV. Non-transduced or 1^(st) and2^(nd) generation CIR transduced T-cells were co-cultured with HIVinfected CEM-SS cells for two days and stained with anti-CD8 andanti-p24gag antibodies. An aliquot of unstained cells were washed andcontinued to culture for another nine days and stained with anti-CD8 andanti-p24gag antibodies. A representative of two experiments is shown. Atday two, infected CD8+ cells show as a distinct population (circled) inthe 1^(st) and 2^(nd) generation T-cells compared to non-transducedcells. There is no distinct population of cells seen in non-Td CD8+cells (day two, upper right quadrant). % modified cells for thisexperiment is: 1^(st) generation=67% and 2^(nd) generation=68%.

FIG. 7 is a graph showing the percent of specific killing as a functionof the ratio of Effector to Target cells. Hege CIR, 1^(st), and 2^(nd)generation T-cells were cultured with ⁵¹Cr labeled uninfected orchronically infected with HIV-1 IIIB CEM-SS cells. Cytotoxicity isdetermined from ⁵¹Cr release to the culture media after 18 hrs ofco-culture at the indicated ratios of Effector to Target, and % specifickilling is calculated as follows:(experimental-control)/(maximal-control)×100. % modified cells for Hegeis 47%, for 1^(St) generation is 25%, and for 2^(nd) generation is 47%.Data shown is representative of two experiments. This is calculated bytaking mean value of CEM-SS control as spontaneousrelease=(Expt-control)/(Max-control).

FIG. 8 is a pair of graphs showing the amount of secretion of theindicated cytokine in the indicate cells types. 1^(st) and 2^(nd)generation T-cells were assayed for IL2 or interferon gamma (IFNγ)secretion by culturing for 24 hrs on anti-CD4 (5 μg/ml) coated plates.Data represented as fold change over 1^(st) generation. IL2 data shownis average±SEM of three experiments. IFN-γ data shown is average±SEM oftwo experiments. % modified cells are similar for 1^(st) and 2^(nd)generation T-cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention features nucleic acid constructs encoding chimeric immuneT-cell receptors (CIRs) that are useful for treating HIV in patients. Ingeneral, the CIRs contain an extracellular domain which targets HIV orHIV-infected cells (e.g., the extracellular domain of CD4), atransmembrane domain, and a cytoplasmic domain for mediating T-cellactivation (e.g., CD3 zeta and/or the partial extracellular domain ofCD28). The invention also features the use of host cells expressing CIRsin the treatment of HIV. When expressed in the host cells, the CIRs canbe engineered to homodimerize, thereby increasing their potency. Thesehost cells can also contain nucleic acid constructs encoding siRNAagainst HIV genes in order to, e.g., disrupt HIV infection of the hostT-cells. The structure of a prior art CIR and the structures of CIRscontaining the transmembrane domain of CD3 zeta and partiallyextracellular domain of CD28 are depicted in FIG. 1.

Extracellular Domains

The CIRs of the invention feature an extracellular domain able tospecifically bind HIV and cells infected with HIV. The HIV protein gp120binds human CD4. Therefore, the extracellular domain of the CIRs of theinvention can include the extracellular domain of CD4 (e.g., human CD4or fragments thereof). Alternatively, the extracellular domain caninclude any binding moiety specific for HIV and cells infected with HIV,including, HIV specific antibodies (e.g., single-chain Fv antibodyfragments that are specific to gp120 or gp41).

The extracellular domain can optionally include a further protein tag,e.g., a c-myc tag (EQKLISEEDL (SEQ ID NO:4) of human origin, at theN-terminus. The c-myc tag does not obstruct CD4 binding to gp120.Inclusion of c-myc in the sFv based-CIR design does not appear to affectCIR function, but can facilitate future study of the construct.

Cytoplasmic Domains

The CIRs of the invention also feature a cytoplasmic domain forsignaling modulating activation of the host T-cells when bound to HIV orHIV-infected cells. Cytoplasmic domains useful for use in the CIRs ofthe invention include CD3 zeta, or fragments thereof, and for thecytoplasmic domain of CD28, or fragments thereof. The invention alsofeatures the fusion of polypeptides derived from multiple extracellulardomains for potentiating activation of T-cells when bound to HIV orHIV-infected cells (e.g., a cytoplasmic domain that includes both activefragments of CD3 zeta and CD28).

Transmembrane Domains

The CIRs of the invention feature transmembrane domains derived fromCD4, CD28, CD3 zeta, or another protein. Furthermore, the transmembranedomain (or the partial extracellular domains, “pEC”) can be engineeredto facilitate homodimerization of the CIRs when expressed in hostT-cells. This can be accomplished, e.g., with the addition orsubstitution of cysteine residues capable of forming disulfide bondswith a paired molecule.

The inclusion of the transmembrane region of the zeta chain or thetransmembrane and partial extracellular domain of CD28 provides thecapability of intermolecular disulfide bonds. CIRs containing thesetransmembrane/partial extracellular domains are predicted to formdisulfide-linked dimers through a cysteine residue located in thetransmembrane of zeta or in the proximal cysteine residue located in thepartial extracellular domain of CD28 (position 123 of CD28), mimickingthe dimer configuration of native zeta and CD28.

siRNA Constructs

The DNA constructs and host cells of the invention also optionallyfeature components to suppress HIV infection of host T-cells. Suchcomponents include siRNA constructs for suppression of HIV replication.These siRNA constructs can be specific for various HIV targets (reviewedin Morris (2006) Gene Ther 13:553-558; Rossi (2006) BiotechniquesSupp1:25-29; Nekhai (2006) Curr Opin Mol Ther 8:52-61; and Cullen (2005)AIDS Rev 7:22-25). One example is an siRNA targeting a highly conservedsequence in an exon common to both tat and rev, has been shown to beeffective to prevent virus expression and replication (See, e.g., SEQ IDNo. 1). In order to prevent HIV infection of host T-cells, the inventionalso features components to decrease expression of T cell coreceptors(e.g., CCR5 and CCR4). Such suppression would be expected to hinderinfection of host T-cells as people with CCR5Δ32 mutation are resistantto HIV infection. The invention also features the inclusion of multiplesiRNA constructs (e.g., constructs against HIV genes and T-cellreceptors used for HIV infection). Here, one siRNA construct can blockinfection and while a second siRNA construct prevents progression ofinfection.

Methods of designing and expressing siRNA constructs are well known inthe art. For example, the siRNA constructs of the invention can utilizelong-hairpin RNA (IhRNA) to express both CCR5 and Tat/Rev siRNAs. Use ofa IhRNA is a viable approach in controlling HIV-1 replication since asingle long transcript can in theory be processed into multiple siRNAs.Multiple targeting can be achieved from a single long-hairpin precursor,suggesting that multiple siRNAs can be processed from the long hairpinsin vivo. The siRNA constructs of the invention can also include apromoter directing expression in host T-cells. Examples of suchpromoters are U6 and tRNA promoters. Expressing shRNAs from tRNApromoters has several advantages, compared to the more commonly used U6and H1 promoters: tRNA promoters are smaller, provide a variety ofoptions, and are typically expressed at lower levels. Smaller promotersmay be desirable in the nucleic acid constructs of the invention tofacilitate inclusion in a vector including a CIR expression construct.An example of a nucleic acid construct containing a CIR and siRNA is setforth in FIG. 2.

shRNA sequences SEQ ID NO: 5 tat/rev shRNA-sense strand5′-GCGGAGACAGCGACGAAGAGC-3′Ref: Scherer, L. J., R. Frank, and J. J. Rossi. 2007. Nucleic Acids Res35: 2620-2628. ccr5 shRNA-sense strand SEQ ID NO: 65′-GCCUGGGAGAGCUGGGGAA-3′ Ref: Ehsani, Mol Titer Epub ahead of print.shRNA within CIR (SEQ ID NO: 7) -Myc-CD4-CD28-zeta-tcaggtggtggcggttcaggcggaggtggetctggcggtggcggatcg                                   Generic linker (G4S)3GCCCGGATAGCTCAGTcGGTAGAGCACAGACTTTAATCTGAGGGTCCAGGGTCAAGTCCCTGTTCGGGCGCCA                        tRNA promoterGCCTGGGAGAGCTGGGGAATTTGTACGTAGTTCCCCAGCTCTCCCAGGCccr5 shRNA sense     shRNA loop   ccr5 shRNA antisenseggtggcagtggctccggaggttcaggaagcggcggtagtgggagc          generic linker (GGSGS)3GCGGAGACAGCGACGAAGAGCCTTCCTGTCAGAGCGGAGACAGCGACGAAGAGCTTTTTGAAtat/rev shRNA sense    shRNA loop  tat/rev shRNA antisense   terminator sequence

Nucleic Acid Constructs

The nucleic acid constructs of the invention are useful for expressingCIRs and siRNA constructs in host T-cells. CIRs and siRNA constructs canbe included in a single nucleic acid construct or multiple nucleic acidconstructs. In order to facilitate transfection of host cells, thenucleic acid construct can be included in a viral vector (e.g., aretroviral vector or adenoviral vector) or be designed to be transfectedinto a host cell via electroporation or chemical means (e.g., using alipid transfection reagent).

Examples of Nucleic Acid Constructs

Myc-CD4-zeta (1^(st )generation (dimer)) SEQ ID NO: 8ATGAACCGGGGAGTCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGCGCTCCTCCCAGCAGCCACTCAGGGAGAGCAGAAGCTGATCTCCGAGGAGG myc (underline)ACCTGAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGACC CD4TGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGATAAAGATTCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGCGCTGACTCAAGAAGAAGCCTTTGGGACCAAGGAAACTTTCCCCTGATCATCAAGAATCTTAAGATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGGACCAGAAGGAGGAGGTGCAATTGCTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACCCTGACCTTGGAGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGGGGTAAAAACATACAGGGGGGGAAGACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGCACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAGGTGGAGTTCAAAATAGACATCGTGGTGCTAGCTTTCCAGAAGGCCTCCAGCATAGTCTATAAGAAAGAGGGGGAACAGGTGGAGTTCTCCTTCCCACTCGCCTTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGGTGGCAGGCGGAGAGGGCTTCCTCCTCCAAGTCTTGGATCACCTTTGACCTGAAGAACAAGGAAGTGTCTGTAAAACGGGTTACCCAGGACCCTAAGCTCCAGATGGGCAAGAAGCTCCCGCTCCACCTCACCCTGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGAAACCTCACCCTGGCCCTTGAAGCGAAAACAGGAAAGTTGCATCAGGAAGTGAACCTGGTGGTGATGAGAGCCACTCAGCTCCAGAAAAATTTGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGCTGAGCTTGAAACTGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAAGGCGGTGTGGGTGCTGAACCCTGAGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGGGACAGGTCCTGCTGGAATCCAACATCAAGGTTCTGCCCACATGGTCCACCCCGGTGCCTAGGCTGGATCCCAAACTCTGCTACCTGCTGGATGG zeta (Underline)AATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACG CCMyc-CD4-CD28-zeta (2^(nd )generation) SEQ ID NO: 9ATGAACCGGGGAGTCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGCGCTCCTCCCAGCAGCCACTCAGGGAGAGCAGAAGCTGATCTCCGAGGAGG myc (underline)ACCTGAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGACC CD4TGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAACCAGATAAAGATTCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCATCCAAGCTGAATGATCGCGCTGACTCAAGAAGAAGCCTTTGGGACCAAGGAAACTTTCCCCTGATCATCAAGAATCTTAAGATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGGACCAGAAGGAGGAGGTGCAATTGCTAGTGTTCGGATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACCCTGACCTTGGAGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGGGGTAAAAACATACAGGGGGGGAAGACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGCACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAGGTGGAGTTCAAAATAGACATCGTGGTGCTAGCTTTCCAGAAGGCCTCCAGCATAGTCTATAAGAAAGAGGGGGAACAGGTGGAGTTCTCCTTCCCACTCGCCTTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGGTGGCAGGCGGAGAGGGCTTCCTCCTCCAAGTCTTGGATCACCTTTGACCTGAAGAACAAGGAAGTGTCTGTAAAACGGGTTACCCAGGACCCTAAGCTCCAGATGGGCAAGAAGCTCCCGCTCCACCTCACCCTGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGAAACCTCACCCTGGCCCTTGAAGCGAAAACAGGAAAGTTGCATCAGGAAGTGAACCTGGTGGTGATGAGAGCCACTCAGCTCCAGAAAAATTTGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGCTGAGCTTGAAACTGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAAGGCGGTGTGGGTGCTGAACCCTGAGGCGGGGATGTGGCAGTGTCTGCTGAGTGACTCGGGACAGGTCCTGCTGGAATCCAACATCAAGGTTCTGCCCACATGGTCCACCCCGGTGCCTAGGAAAATTGAAGTTATGTATCCTCCTCCTTAC CD28 (underline)CTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTG zetaAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGAC GCC

The amino acid sequence for the CD28 and CD3 zeta portions of the 2^(nd)generation construct is

(SEQ ID NO: 10) K I E V Met Y P P P Y L D N E K S N G T I I H V K G K H L C P S P L F P G P S K PF W V L V V V G G VL A C Y S L L V T V A F I I F W V R S K R S R L LH S D Y Met N Met T P R R P G P T R K H Y Q P Y A P P R D F A A Y R S R V K F S R S A D AP A Y Q Q GQ N Q L Y N E L N L G R R E E Y D V L D K R R G RD P E Met G G KP R R K N P Q E G L Y N E L Q K D K Met A E A Y S E I G Met K G E R R R G K GH D G LY Q G L S T A T K D T Y D A

Host T-Cells

The host T-cells of the invention can be isolated from, e.g., a patientinfected with HIV. The host T-cells are transfected or infected withnucleic acid constructs of the invention (e.g., nucleic acid constructsencoding a CIR and, optionally, one or more siRNA constructs). Prior toadministration to a patient the T-cells can be expanded in cell culture.In one embodiment, the modified T-cells are administered to the patientfrom whom they were originally isolated.

In one embodiment, PBMCs are isolated by standard techniques andtransduced with a CIR. Cells are administered to the patient in a doseof between 10⁹ and 10¹⁰ cells (e.g., 10⁹, 5×10⁹, or 10¹⁰ cells). Cellscan be isolated once and expanded for multiple administrations or aseparate isolation and transduction can be performed with each round oftreatment.

Treatment can be, e.g., a single treatment, monthly treatment,semi-annual treatment, or annual treatment.

Additional Agents

Additional antiviral can be, for example, a protease inhibitor, areverse transcriptase inhibitor, an integrase inhibitor, a CCR5antagonist, a fusion inhibitor, or a second maturation inhibitor. Theadditional antiviral agent can be, without limitation, azidovudine(AZT), didanosine (dideoxyinosine, ddI), d4T, zalcitabine(dideoxycytosine, ddC), nevirapine, lamivudine (epivir, 3TC), saquinavir(Invirase), ritonavir (Norvir), indinavir (Crixivan), and delavirdine(Rescriptor).

Additional Therapies

The methods of the invention can be combined with, e.g., lymphodepletionprior to administration of host T-cells. Furthermore, treatment can alsoinclude the administration of one or more cytokines, e.g., IL-2, IL-7,and IL-15.

Experimental Results

Construction of Retroviral Vectors

The chimeric immune T-cell receptor (CIR) of the prior anti-HIV designerT cell trials had the structure of extracellular domain of CD4 (apolypeptide corresponding to amino acids 1-372 of SEQ ID NO:1),transmembrane domain of CD4 (a polypeptide corresponding to amino acids373-395 of SEQ ID NO:1) and cytoplasmic domain of zeta (a polypeptidecorresponding to amino acids 31-142 of SEQ ID NO:3) (Deeks et al. (2002)Mol Ther 5:788-797, Mitsuyasu et al. (2000) Blood 96:785-793) (herein“Hege CIR”, FIG. 1). We also designed a signal one-only CIR that issimilar to the Hege CIR except that the transmembrane domain of zeta (apolypeptide corresponding to amino acids residues 7-30 of SEQ ID NO:3)was substituted (1^(st) generation CIR, FIG. 1). Lastly, we created aconstruct that integrates CD28 as well as zeta signaling in a two signalformat (2^(nd) generation CIR, FIG. 1). This employs the sameextracellular domain of CD4 (a polypeptide corresponding to amino acids1-372 of SEQ ID NO:1) with a partial extracellular doman/transmembranedomain/cytoplasmic domain of CD28 (a polypeptide corresponding to aminoacids 127-234 of SEQ ID NO:2) and is expressed as dimer.

In addition, a c-myc tag (EQKLISEEDL) of human origin is also includedin our constructs at the N-terminus of CD4.

1^(st) and 2^(nd) generation CIRs were constructed in the MFG retrovirusvector. Retrovirus was created by ping pong between the E+86 ecotropicand PG13 amphotropic cell lines. PG13 is a helper cell line derived frommurine fibroblasts that is used to create vector producer cells (VPC)for retroviral production. VPCs were sorted for the highest transgeneexpression and viral supernatants were harvested as described Beaudoinet al. ((2008) J Virol Methods 148:253-259).

Expression of CIRs

Viral supernatants from PG13 VPCs were used to transduce human PBMCs.PBMCs from normal healthy individuals were purified and activated withanti-CD3 antibody (OKT3) and 100 U/ml IL2 for two days and transducedwith retrovirus by spinoculation on a retronectin coated plate. Wedetermined the surface expression of CIRs on CD8+ T-cells by doublestaining for CD8 and CD4 and determined the transduction rate (as %modified cells, FIG. 3). Transduction of activated human T-cellsroutinely yield 40 to 70% transduction rates with these anti-HIV CIRs.

We co-cultured transduced or non-transduced T-cells with CEM-SSHIV+(chronically infected with HIV-1 IIIB) or HIV− cells (at an E:Tratio of 1:1 or 1:10). We determined the presence of transduced cells inthe culture by staining with anti-CD4 and anti-CD8 antibodies.Co-culture of Hege CIR T-cells with CEM-SS HIV+ cells at an E:T ratio of1:10 induced cell death and all the Hege CIR cells disappeared from theculture by day 3 (FIG. 4). At an E:T ratio of 1:1, Hege CIR T cells werestill present in the culture at day 13, with killing of all target cellsin the culture (observed by flow cytometry analysis). Cell deathobserved in the Hege CIR designer T-cells could be either due toheightened sensitivity to Activation Induced Cell Death (AICD) or to HIVinfection. To test this, Hege CIR cells were treated withanti-retroviral drug AZT and co-cultured with HIV infected CEM-SS cells.AZT treated Hege CIR cells did not die when co-cultured with highertarget ratio (1:10, FIG. 5). These data suggest that Hege CIR cellsbecome infected with HIV and die by either HIV induced apoptosis orkilled by other CIR containing T-cells (fratricide). These data suggestand we hypothesize that one of the reasons for the failure of Hege CIRin the clinical trials could be due to highest susceptibility to HIVinfection and elimination from the patients.

Susceptibility of Designer T-Cells to HIV Infection

HIV infects CD4+ T-cells by binding to CD4 receptor and a co-receptor(CXCR4 or CCR5). Since CIR has an extracellular CD4 domain, wepostulated that this could be used by HIV to infect all CIR+ T-cells,including CD8+ T-cells. HIV infection of CIR+CD8+ cells was determinedby co-culturing CIR containing T-cells with HIV+ CEM-SS cells andstaining for p24-gag antigen, an indicator of productive infection.After two days of culture, cells were stained with anti-CD8 and anti-p24gag antibodies. In contrast to non-transduced cultures, CD8+ cells areinfected with HIV in transduced cultures (1^(st) and 2^(nd) generation)(FIG. 6). At day 11 we did not detect any HIV infected CD8+ cells ineither 1⁴ or 2^(nd) generation CIR containing T-cells (FIG. 6).

Killing of HIV-Infected Cells by Modified T-Cells.

In order to compare the potencies of Hege CIR, 1^(st), and 2^(nd)generation anti-HIV CIR in killing of target cells, activated T-cellswere transduced as described above. Target cells (HIV-infected oruninfected CEM-SS cells) were labeled with ⁵¹Cr for 5 hrs (50 μCi for1×10⁶ cells) and co-cultured with transduced T-cells at indicated E:Tratios for 18 hrs. Hege CIR T-cells and our 1^(st) and 2^(nd) generationdesigner T-cells were all equally potent in killing HIV+ target cells(FIG. 7).

Cytokine Secretion by Modified T-Cells.

Human T-cells transduced with 1^(st) and 2^(nd) generation CIRcontaining T-cells were tested for their ability to secrete cytokinesupon stimulation through the CIR. Transduced or non-transduced T-cellswere cultured on anti-CD4 antibody coated plates for 24 hrs. IL2secretion was measured with an ELISA kit. 2^(nd) generation T-cellsproduced more IL2 than 1^(st) generation T-cells when stimulated withanti-CD4 antibody (FIG. 8). In contrast, IFNγ secretion is similar withanti-CD4 stimulation of 1^(st) and 2^(nd) generation designer T-cells,as is typical for T cell signaling.

Conferring Resistance of Designer T-Cells to HIV Infection

HIV infects CD4+ T-cells by binding to CD4 receptor and a co-receptor(CXCR4 or CCR5). As shown above, CD8+CIR+ cells (1^(st) and 2^(nd)generation) are susceptible to HIV infection (day two, FIG. 6). At day11 we did not detect any HIV infected CD8+ cells in either 1^(st) or2^(nd) generation T-cells (FIG. 6). These data suggest that modifiedT-cells could kill other HIV infected modified T-cells. Nevertheless,this is a potential source of loss of effector cells to combat HIV andprovides a new reservoir to increase patient HIV load. It is thereforebecomes important to eliminate or reduce the potential for HIV to infectmodified T-cells.

Other Embodiments

Various modifications and variations of the described methods andcompositions of the invention will be apparent to those skilled in theart without departing from the scope and spirit of the invention.Although the invention has been described in connection with specificdesired embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention that are obvious to those skilled in the fields ofmedicine, immunology, pharmacology, endocrinology, or related fields areintended to be within the scope of the invention.

All publications mentioned in this specification are herein incorporatedby reference to the same extent as if each independent publication wasspecifically and individually incorporated by reference.

What is claimed is:
 1. A nucleic acid construct encoding a chimericprotein comprising (i) an extracellular domain of CD4, or a fragmentthereof, (ii) a transmembrane domain, and (iii) a cytoplasmic domaincomprising a) the cytoplasmic domain of the CD3 zeta chain, or afragment thereof and b) the cytoplasmic domain of CD28, or a fragmentthereof.
 2. The nucleic acid construct of claim 1, wherein said chimericprotein is capable of forming a homodimer when expressed in a T cell. 3.The nucleic acid construct of claim 2, wherein the dimerized chimericproteins are capable of forming at least one disulfide bond.
 4. Thenucleic acid construct of claim 1, wherein said transmembrane domaincomprises a polypeptide selected from the group consisting of thetransmembrane domain of the CD3 zeta chain and the transmembrane domainof CD28.
 5. The nucleic acid construct of claim 4, wherein saidtransmembrane domain comprises amino acids 7-30 of SEQ 10 NO:3. 6.(canceled)
 7. The nucleic acid construct of claim 1, wherein saidextracellular domain of CD4 comprises amino acids 1-372 of SEQ 10 NO:1.8. The nucleic acid construct of claim 1, wherein said cytoplasmicdomain of the CD3 zeta chain comprises amino acids 31-142 of SEQ 10NO:3.
 9. The nucleic acid construct of claim 1, wherein said cytoplasmicdomain of CD28 comprises amino acids 127-234 of SEQ 10 NO:2.
 10. Thenucleic acid construct of claim 1, wherein said cytoplasmic domaincomprises the amino acid sequence of SEQ ID NO:10.
 11. A nucleic acidconstruct encoding a chimeric protein comprising (i) an extracellulardomain of CD4, or a fragment thereof, (ii) a transmembrane domain, and(iii) a cytoplasmic domain of the CD3 zeta chain, or a fragment thereof,wherein said chimeric protein is capable of forming a homodimer whenexpressed in a T cell.
 12. The nucleic acid construct of claim 11,wherein the chimeric protein, when in a homodimer, is capable of formingat least one disulfide bond with the chimeric protein with which it isdimerized.
 13. The nucleic acid construct of claim 11, wherein saidtransmembrane domain comprises the transmembrane domain of the CD3 zetachain or the transmembrane domain of CD28.
 14. The nucleic acidconstruct of claim 13, wherein said transmembrane domain comprises aminoacids 7-30 of SEQ ID NO:3.
 15. (canceled)
 16. The nucleic acid constructof claim 11, wherein said extracellular domain of CD4 comprises aminoacids 1-372 of SEQ ID NO:1.
 17. The nucleic acid construct of claim 11,wherein said cytoplasmic domain of the CD3 zeta chain comprises aminoacids 31-142 of SEQ ID NO:3.
 18. (canceled)
 19. (canceled) 20.(canceled)
 21. (canceled)
 22. (canceled)
 23. (canceled)
 24. (canceled)25. A host cell comprising the nucleic acid construct of claim 1 and anucleic acid construct encoding an siRNA.
 26. The host cell of claim 25,wherein said siRNA is against CCR5.
 27. The host cell of claim 25,wherein said siRNA is against Tat/Rev.
 28. A method of treating apatient infected with HIV by administering a composition comprising hostcell of claim
 22. 29. (canceled)